Lab 1 Prep: Carb metabolism and glucose tolerance

VET30050: Carbohydrate Metabolism and Glucose Tolerance

Experiment 1: Ability of Tissues to Release Glucose into the Circulation

Principle

• Tissues are supplied with carbohydrates in the form of glucose transported in plasma.

  • Plasma-derived glucose sources include:

  • Small intestinal absorption.

  • Release from cells after dephosphorylation of glucose-6-phosphate following:

    • Glycogenolysis.

    • Gluconeogenesis.

• The release of free glucose by cells is contingent on their content of glucose-6-phosphatase.

  • Glucose-6-phosphatase is essential for hydrolyzing glucose-6-phosphate into free glucose.

• Tissues to be analyzed for glucose-6-phosphatase content:

  • Liver.

  • Kidney.

  • Cardiac muscle (heart).

  • Skeletal muscle.

• Measurement of free glucose or inorganic phosphate provides an index of tissue capacity to contribute free glucose to plasma.

• Measurement methodology involves:

  1. Step 1: Glucose Oxidation by Glucose Oxidase (GOD)

  • Reaction: $ext{GLUCOSE} + ext{O}<em>2 + ext{H}</em>2 ext{O} <br>ightarrow ext{GLUCONATE} + ext{H}<em>2 ext{O}</em>2$

  1. Step 2: Reaction of Hydrogen Peroxide by Peroxidase (POD)

  • Reaction: $2 ext{H}<em>2 ext{O}</em>2 + 4- ext{Aminophenazone} + ext{Phenol} <br>ightarrow ext{4-(p-Benzoquinone-mono-imino)phenazone (coloured compound)} + 4 ext{H}_2 ext{O}$

Materials

• Homogenates of:

  • Rat liver (L).

  • Cardiac muscle (CM).

  • Skeletal muscle (SK).

  • Kidney (K).

• Glucose-6-phosphate (G6P): 10 mM (di-sodium salt) in citrate buffer (pH 6.8, 200 mM).

Learning Outcomes

• Understand mechanisms controlling blood glucose levels and effects of disturbances in these mechanisms.

• Learn about the relationship between triacylglycerol and non-esterified fatty acid (NEFA) levels in blood under different dietary conditions.

Experiment 2: Assessment of Insulin-Secreting Capacity of the Pancreas (Glucose Tolerance Test)

Rationale

• To account for variations due to the oral glucose tolerance test, utilize the intravenous glucose tolerance test.

• Glucose measurement follows the methodology established in Experiment 1.

Procedure

1. Blood Samples":

   • Control sample (pre-glucose loading) taken at -15 min.

2. Glucose Loading:

   • Administer a 50% w/v glucose solution (2.8 M) intravenously at 1 g/kg (5.55 mmol) for 1 minute (0 min timepoint).

3. Subsequent Sampling:

   • Blood samples taken at:

     • 0 min (immediately after loading).

     • Every 15 minutes for the next 90 minutes (-15, 0, +15, +30, +45, +60, +90 min).

4. Measurement Supplies:

   • Two blood samples provided for glucose measurement from different time points.

   • Glucose standard 5.55 mM (GSTD) and Glucose Reagent (GR) with GOD and POD.

Measurement Reaction

• Same as in Experiment 1:

  • Step 1: Reaction with glucose oxidase:

  • $ext{GLUCOSE} + ext{O}<em>2 + ext{H}</em>2 ext{O} <br>ightarrow ext{GLUCONATE} + ext{H}<em>2 ext{O}</em>2$

  • Step 2: Reaction by peroxidase:

  • $2 ext{H}<em>2 ext{O}</em>2 + 4- ext{Aminophenazone} + ext{Phenol} <br>ightarrow 4-(p- ext{Benzoquinone-mono-imino}) ext{phenazone (coloured compound)} + 4 ext{H}_2 ext{O}$

Procedure Steps

• Similar to Experiment 1 with adjusted reagents and biological samples.

Experiment 3: Lipid Metabolism

Background

• Classification of Lipids:

  • Structural lipids: Integral parts of cells/tissues.

  • Depot lipids: Major energy storage.

• Lipid formation and degradation occur continuously, with flux of molecules through processes determined by:

  • Anabolic/catabolic metabolism.

  • Metabolic diseases (e.g., diabetes mellitus).

  • Exercise phases.

• Lipid concentrations in tissues are influenced by:

  • Rate of transport into cells.

  • Rate of synthesis.

  • Rate of lipolysis and oxidation.

• Hormonal control affects metabolite levels and enzyme activities.

• Notably, animals can synthesize lipid variants from carbohydrates.

• Ruminants specifically process dietary lipids pre-absorption.

Comparison Table 1: Values of Fatty Acids in Various Sources

Experiments 3A and 3B: Variations of Serum Triglyceride Levels

Principle

• Triglyceride Measurement:

  • Triglycerides react to give fatty acids and glycerol.

  • Glycerol is then converted to glycerol-3-phosphate and subsequently oxidized, generating a dye whose absorbance is measured at 540nm.

Procedure for 3A

1. Add 190 μl of triglyceride reagent to six wells.

2. In the first well, add 10 μl of standard.

3. In the following four wells, add 10 μl of serum samples (from:

   • Well-fed dog.

   • Dog fasted for 24h.

   • Well-fed sheep.

   • Sheep fasted for 48h).

4. Final well is blank with 10 μl of water.

5. Measure absorbance at 540nm.

Procedure for 3B: NEFA Level Variations

• Use absorbance values from previous analysis to calculate NEFA concentrations:

• Calculation:

  • $ext{Concentration of NEFA (mM)} = rac{ ext{Absorbance of Unknown} imes ext{Concentration of Standard (1.0 mM)}}{ ext{Absorbance of Standard}}$

Experiment 4: Effects of Metabolic Status on Urine Composition in Dogs

Procedure

• Analyze urine samples (U1 to U4) using “Clinistix” test strips for:

  • pH levels.

  • Glucose concentration.

  • Levels of ketone bodies.
    | Dog Urine Sample | pH | Glucose | Ketones |
    |------------------|----|---------|---------|
    | U1 (small meal) | | | |
    | U2 (large meal) | | | |
    | U3 (diabetic) | | | |
    | U4 (fasting) | | | |

Expectation Discussion

• Reflect on whether results align with expected outcomes based on metabolic state.

Laboratory Report Instructions

• Subtract baseline absorption of blank from readings for standard and unknown samples.

Calculations for Experiment 1

• 
  

• Calculate glucose concentration from absorbance values using the formula:

  • $ext{Concentration of Plasma Sample (mM)} = rac{ ext{Absorbance of Plasma Sample} imes ext{Concentration of Standard (5.55mM)}}{ ext{Absorbance of Standard (S)}}$

• 
  

• Record glucose concentrations at indicated time points for analysis:
  

  Time (min)	Absorbance	Glucose Concentration (mM)
  -15
  0
  +15
  +30
  +45
  +60
  +90
  Standard	5.5
  Graphical Analysis

  • Plot blood glucose concentration (mM) against time (min).

  • Compare experimental data to expected glucose tolerance curve profile from a diabetic animal.

  Variations in Triglyceride and NEFA Levels

  Data Summary

  Treatment	Serum Triacylglycerol Absorbance	Concentration (mM)	Serum NEFA Absorbance	Concentration (mM)
  1. Dog: Well Fed	0.26
  2. Dog: Fasted 24h	0.42
  3. Sheep: Well Fed	0.29
  4. Sheep: Fasted 48h	0.36
  Standards	3.0		0.30	1.0

  Analysis Questions

  • Discuss the factors leading to variations observed in triglyceride and NEFA levels.

  • Compare lipid metabolism alterations during periods of prolonged starvation and in diabetes mellitus.

  Additional Questions

  • Discuss the role of ruminal microbes in protein metabolism in ruminants.

  • Evaluate disadvantages of not providing good quality protein to ruminants.